H, 


{  .  S.  DEPARTMENT    Ol 

BUREAU  OF  PLANT  INDUSTRY-  BUL: 


B.  T.  (.  11.1.1'HAV,  Chiei 


AGRICULTURE. 

NO.  72,  PARI  IV. 


,/■  /SG-i. 


INOCULATION  OF  S 


NITROGEN-FIXING  BA 


BY 


A.   F.  WOODS, 
Acting  Chief  op  the  Bureai   oi  Plant  Industry. 


WASHINGTON: 

GOVERNMENT     PRINTING    OFFICE. 
1905. 


B.  I".   I—  KJ3. 


INOCULATION  OF  SOIL  WITH  NITROGEN- 
FIXING  BACTERIA. 


INTRODUCTION. 

The  publication  of  the.  results  obtained  with  pure  cultures  in  inocu- 
lating leguminous  plants  has  resulted  in  a  very  greal  demand  being 
made  upon  the  Department  of  Agriculture  for  inoculating  material. 
The  distribution  made  during  L904  was  for  the  purpose  of  obtaining  ;i 
large  number  of  tests  of  the  method  under  average  farm  condition-, 
and  it  was  impossible  to  anticipate  the  demand  which  has  arisen  this 
spring  (1905),  the  total  quantity  prepared  for  spring  distribution 
having  been  promised  early  in  February.  It  is  expected,  however, 
that  thi-  fall  and  next  springs  further  distribution  will  be  made  as  far 
as  our  limited  facilities  will  permit.  Statements  to  the  effeel  that  the 
Depart inent  has  -topped  the  distribution  of  these  culture-,  are  therefore 
erroneous.  Applications  for  future  distributions  should  state  what 
legume  is  to  be  ■-own.  time  of  --oh  ing,  and  quantity  of  seed  to  be  treated. 

THE  COMMERCIAL,  PRODUCTION  OF  CULTURES. 

The  patent  which  the  Department  of  Agriculture  holds  upon  the 
met  hod  of  grow  ing  and  disl  ributing  these  organisms  was  taken  out  in 

such  a  way  that  no i  can  maintain  a  monopoly  of  the  manufacture 

of  9uch  culture-,     li    is   held  in  the  name  of  Dr.  George  /.  M c, 

who  developed  and  perfected  the  met li.nl.  a-  described  in  former 
publications.  Upon  application  the  Department  furnishes  without 
discrimination  all  necessary  information,  and  a-  far  a-  possible  "  start- 
ing" or  Eoundat  ion  culture--,  to  the  bacteriologists  representing  experi- 
ment stations  and  commercial  concerns  uhieh  claim  to  1m-  properly 
equipped,  Gut  it  doe-  not  in  any  wa\  guarantee  their  product.  It  is 
not  likelj  that  persons  without  expert  knowledge  can  successfully 
multiply  cultures  <>i'  these  organisms  for  -ale  or  distribution,  and  it 
is  understood  thai  an\  cultures  furnished  are  to  he  treated  according 
to  the  methods  devised  l>_\  tin    Department. 

3 


4  NITROGEN-FIXING    BACTERIA. 

Before  experimenting  with  any  bacterial  preparations  for  legumes, 
the  farmer  should  study  thoroughly  the  soil  conditions  under  which 
the  use  of  cultures  offers  any  possibility  of  gain." 

Briefly,  these  conditions  may  be  summed  up  as  follows: 

WHEN  INOCULATION  IS  NECESSARY. 

Inoculation  is  necessary — 

(1)  On  a  soil  low  in  organic  matter  that  has  not  previously  borne 
leguminous  crops. 

(2)  If  the  legumes  previously  grown  on  the  same  land  were  devoid 
of  nodules,  or  "  nitrogen  knots,"  showing  the  need  for  supplying  the 
nodule-forming  bacteria. 

(3)  When  the  legume  to  be  sown  belongs  to  a  species  not  closely 
related  to  one  previously  grown  on  the  same  soil.  For  instance,  soil 
in  which  red  clover  forms  nodules  will  often  fail  to  produce  nodules 
on  alfalfa  when  sown  with  alfalfa  for  the  first  time. 

WHEN   INOCULATION   MAY   PROVE   ADVANTAGEOUS. 

Inoculation  may  prove  advantageous — 

(1)  When  the  soil  produces  a  sickly  growth  of  legumes,  even  though 
their  roots  show  some  nodules. 

If  the  cultures  introduced  are  of  the  highest  virility,  their  use  will 
often  result  in  a  more  vigorous  growth. 

(2)  When  a  leguminous  crop  already  sown  has  made  a  stand,  but 
gives  evidence  of  failing,  due  to  the  absence  of  root  nodules. 

The  use  of  the  culture  liquid  as  a  spray  or  by  mixture  with  soil  and 
top-dressing  may  save  the  stand  if  other  conditions  are  favorable. 

WHEN   INOCULATION   IS   UNNECESSARY. 

On  the  other  hand,  inoculation  is  unnecessary  and  offers  little  jwos- 
■pect  of  gain — 

(1)  Where  the  leguminous  crops  usually  grown  arc  producing  up  to 
the  average  and  the  roots  show  nodules  in  normal  abundance. 

Cultures  of  nitrogen-fixing  bacteria  are  not  to  In  regarded  in  the  light 
of  fertilizers,  increasing  yields  under  all  average  conditions.  They 
do  not  contain  the  nitrogen  itself,  but  the  bacteria  make  it  possible 
for  the  legumes  to  secure  nitrogen  from  the  air  (through  the  forma- 
tion of  root  nodules),  and  where  the  soil  is  already  adequately  supplied 
with  these  bacteria  it  will  not  usually  pay  to  practice  any  form  of 
artificial  inoculation. 

(2)  When  the  soil  is  already  rich  in  nitrogen. 

It  is  neither  necessary  nor  profitable  to  inoculate  a  soil  rich  in  nitro- 
gen when  sowing  legumes.     Not  only  does  the  available  nitrogen  in 

«  Fully  described  in  Farmers'  Bulletin  No.  214  of  the  Department  of  Agriculture, 
which  will  be  sent  without  cost  upon  application  to  the  Secretary  of  Agriculture. 


COST    OF    <  I  1.111:1  I) 

the  soil  render  the  formation  of  nodules  less  necessary,  bul  nit  rogenous 
materials  in  the  soil  largely  prei » ■  1 1 1  the  bacteria  from  forming  nodules. 
Am  increased  virility  in  nitrogen-fixing  power  possessed  by  any 
t\  pes  of  bacteria  yel  distributed  may  be  rapidlj  losl  in  a  soil  contain- 
ing an  abundance  of  nitrogen,  because  the  bacteria  are  rapidly  multi- 
plying in  a  medium  in  which  there  is  no  premium  on  vigor  in  securing 
atmospheric  nitrogen. 

WHEN  FAILURE  IS  TO  BE  EXPECTED. 

Inoculation  will  fail  where  other  conditions  (aside  from  the  need  of 
bacteria)  are  not  taken  into  account,  as  the  following: 

(1)  In -oil  thai  is  acid  and  in  need. of  lime. 

Liming  to  correct  acidity  is  as  important  for  the  proper  activity  of 
the  bacteria  as  for  the  growth  of  the  plants. 

(2)  En  soil  that  responds  in  a  marked  way  to  fertilizers,  such  as 
potash,  phosphoric  acid,  or  lime. 

The  activity  of  the  bacteria  in  securing  nitrogen  from  the  air  ami 
rendering  it  available  to  the  legumes  doe-  not  do  away  with  the  need 
for  Such  fertilizing  elements  as  potash  and  phosphorus. 

(3)  It  must  also  be  remembered  that  inoculation  does  not  "act  like 
magic;"  it  will  not  overcome  results  due  to  had  seed,  improper 
preparation  and  cultivation  of  ground,  and  decidedly  adverse  condi- 
tion- of  w  eat  her  or  climate. 

In  the  use  of  cultures,  also,  failure  is  a  hue- 1  certain  where  the  direc- 
tions are  not  carefully  studied  and  intelligently  followed. 

(4)  As  the  physics,  the  chemistry,  and  the  biolog}  of  soils  are 
studied  in  the  laboratory  and  by  means  of  actual  field-plot  trial-  to 
determine  yield  and  quality  of  crops  and  the  effect  of  one  crop  on  the 
following  crop-,  the  ven  great  complexity  of  soil  and  farm  manage- 
ment becomes  more  manifest. 

The  value  of  pure-bred  bacteria,  whether  associated  with  the  crop 
or  existing  independently  in  the  soil,  as  is  true  of  fertilizers,  can  not 
lie  predicted  u  ith  certainty  on  any  soil  \\  ithout  trial.  Success  on  simi- 
lar near-by  lands  may  he  taken  as  good  evidence.  But,  unlike  fer- 
tilizer-, bacteria  should  in  time  We  so  inexpensive  that  each  farmer 
can  afford  to  try  them  for  each  leguminous  crop  on  each  field  or  soil 
t\  pe  on  hi-  farm.  The  method-  of  distributing  in  dried  form  and  the 
easy  methods  of  multiplying  on  the  farm  in  sufficient  quantities  to 
inoculate  fields  will  make  it  possible  to  have  all  fields  inoculated  at  all 
time-. 

COST    OF    CULTURES. 

The  question  of  the  proper  price  for  the  commercial  product  is  caus- 
ing considerable  inquirj  among  prospective  experimenters  and  i-  of 

importance.     The  expenses  which  a  commercial  concern  must  neces- 
sarily  meet,  such  a-  rent,  heat,  light,  insurance,  postage,  advertising, 


6  NITROGEN-FIXING    BACTERIA. 

etc. ,  aside  from  laboratory  assistance  and  clerical  hire,  make  airy  com- 
parison with  the  cost  to  the  Government  of  similar  cultures  difficult. 
The  statement  that  the  cultures  cost  but  a  few  cents  an  acre  refers 
only  to  the  raw  materials  which  make  up  the  package.  It  is  more  than 
probable  that  natural  competition  will  considerably  reduce  the  present 
valuation  of  the  commercial  product,  and  the  wisdom  of  patenting 
the  Department's  methods  to  prevent  the  formation  of  a  monopoly  is 
already  demonstrated. 

INCREASING  CULTURES. 

We  are  receiving  numerous  requests  from  persons  who  have  secured 
commercial  cultures,  as  well  as  those  sent  out  from  the  Department  of 
Agriculture,  for  information  as  to  the  methods  employed  in  producing 
a  large  quantity  of  liquid  culture  from  the  dry  culture  secured  as  a 
starter;  that  is,  how  to  make  an  "acre  culture'1  do  for  25  or  100 
acres.  Such  methods  will  give  good  results  only  when  special  pre- 
cautions are  taken,  and  on  this  account  have  not  been  generally  recom- 
mended. The  contaminations,  such  as  yeasts,  molds,  etc.,  which  are 
bound  to  occur  to  a  greater  or  less  extent,  are  apt  to  take  possession 
of  the  culture  solution  in  which  the  bacteria  are  being  multiplied,  and 
unless  great  care  is  taken  in  thoroughly  sterilizing  all  utensils  em- 
ployed the  resulting  culture  will  have  no  beneficial  effect.  The  extra 
time  required  to  secure  sufficient  growth  of  bacteria  in  10  gallons  of 
solution  from  a  dry  culture  originally  intended  to  produce  a  1-gallou 
liquid  culture  makes  the  risk  from  contamination  much  greater  than 
where  the  dry  culture  is  proportioned  in  size  to  the  larger  amount  of 
solution.  If  a  growth  sufficient  to  cloud  the  solution  takes  place 
within  two  days,  the  chances  of  securing  an  efficient  culture  are  much 
better  than  where  a  longer  time  is  taken;  so  that  the  volume  of  solution 
prepared  should  never  exceed  the  actual  requirements  of  the  occasion. 

The  following  directions  are  based  on  making  10  gallons  of  liquid 
culture,  sufficient  to  inoculate  20  bushels  of  seed.  By  a  little  compu- 
tation the  directions  may  be  adapted  to  5  gallons  or  to  any  intermedi- 
ate quantities. 

PREPARING  AND  USING  THE  CULTURE  SOLUTION. 

To  prepare  the  culture  solution,  first  select  the  tub,  bucket,  or  other 
vessel  in  which  you  wish  to  grow  the  bacteria.  Clean  and  scald  it  out 
thoroughly.  For  making  the  culture  solution,  rain  water  that  has 
been  thoroughly  boiled  and  allowed  to  cool  is  best,  though  any  good 
drinking  water  will  answer.  Add  to  10  gallons  of  water  12  ounces  of 
either  brown  or  granulated  (preferably  granulated)  sugar,  14  ounces 
of  potassium  phosphate  (monobasic),  which  can  be  obtained  at  any 
drug  store,  and  one-sixteenth  ounce  (30 grains) of  magnesium  sulphate. 


* 


KEEPING    CI   LTURES    FOR    FUT1   R]      USE.  ( 

Stir  until  dissolved,  then  carefully  open  the  small  package  containing 
the  bacteria-laden  cotton  and  drop  the  cotton  into  the  solution.  I><> 
doI  handle  any  more  than  is  absolutely  necessary.  Cover  the  tub 
with  a  moi  -i ,  clean  cloth  toproteci  from  dust,  mold  spores,  etc.  Keep 
in  a  warm  place,  but  never  let  the  temperature  rise  above  Mood  heat. 
A.fter  twenty  four  hours  add  6  ounces  of  ammonium  phosphate  and 
allow  the  mixture  to  -land  for  another  twenty  four  hours.  The  Liquid 
should  now  be  cloudy  and  ready  for  use;  if  sufficient  growth  has  not 
taken  place  to  bring  about  this  cloudiness,  further  tune  should  be 
given,  not  to  exceed  .1  few  daj  s. 

To  inoculate  seed.  Use  enough  culture  liquid  to  moisten  the  seed 
thoroughly  aboul  one  half  of  a  gallon  per  bushel.  This  inoculating 
may  he  done  either  in  a  tub  ^v  trough,  or  by  sprinkling  the  culture 
liquid  on  the  seed  on  a  clean  floor  and  stirring  and  turning  the  heaps 
of  seed  with  shovels  until  all  are  thoroughly  moistened.  After  inoc 
ulation  the  seed  should  he  spread  out  in  a  clean,  shady  plaee  until 
sufficiently  dry  to  handle.  If  planting  is  not  to  he  done  at  once,  the 
seed  must  he  thoroughly  dried  to  prevent  molding.  In  dry  weather 
aboul  25  bushels  can  he  dried  in  half  a  day  on  .'inn  square  feet  of  floor 

spaee.      To   do   this   there    must    lie   several    open    windows  or  doors  to 

allow  a  free  circulation  of  air,  and  the  seed  must  be  frequently  stirred 
with  a  lawn  rake.  The  inoculated  seed,  if  thoroughly  dried,  may 
usually  be  kept  without  deterioration  for  several  months. 

'/',,  inoculah  soil.  Take  enough  dry  earth  or  sand  so  that  the  solu- 
tion will    merely  inoi-ten  it.      The   -nil    should    he   preferably  from  the 

field  to  he  inoculated,  so  a-  to  avoid  spreading  diseases  or  weed-. 
Mix  thoroughly,  so  that  all  the  particles  of  soil  are  moistened. 
Thoroughly  mix  this  earth  with  four  or  five  times  as  much;  spread 
this  inoculated  -oil  thinly  and  evenly  over  the  prepared  ground 
exactly  a-  if  spreading  fertilizer.  The  inoculated  soil  should  he  har- 
rowed in  immediately  to  protect  the  bacteria  from  sunlight.  In  using 
this  method  allow  l  gallon  of  the  Liquid  culture  to  1  acre-  or  less. 

Either  <'\'  the  methods  described  may  he  used,  a-  may  he  most 
convenient. 

'/'■■  prevent  any  possible  delay,  tfu  necessary  chemicals  should  he 
ordered  in  advanc< .  If  the  local  druggist  does  not  have  them  in  stock, 
he  .-an  doubtless  secure  them  within  a  reasonable  time. 

KEEPING  CULTURES  FOR  FUTURE  USE. 

The  question  is  frequently  arising  as  to  the  possibility  of  the  farm- 
er'- keeping  over  cultures  from  one  year  to  another  by  soaking  up  a 
little  of  the  lit  pi  id  culture  in  cotton  and  drying  this  coi  1 1  m.  Tins  pro- 
posed  practici  is  not  tobt  advised  hi  any  case.  Contaminations  take 
place  so  readily,  an  tarted  spread  so  rapidly,  that  for  assured 


8  NITROGEN-FIXING     BACTERIA. 

good  results  it  is  absolutely  necessary  to  start  with  a  pure  culture. 
The  pure  culture,  moreover,  can  only  be  prepared  by  a  trained  bac- 
teriologist with  laboratory  facilities.  These  cultures  in  the  dry  state 
will  keep,  under  ordinary  conditions,  from  six  months  to  a  year. 

There  is  an  additional  reason,  fully  as  important,  which  makes  the 
above  method  impracticable.  The  cultivation  of  the  bacteria  for  any 
considerable  length  of  time  in  solutions  containing  ammonium  salts 
rapidly  lessens  their  infective  power  and  their  ability  to  gather  nitro- 
gen from  the  air,  so  that  transfers  or  new  cultures  made  with  absorb- 
ent cotton  from  the  cultures  prepared  for  field  use  would  contain 
organisms  of  reduced  efficiency.  It  is  partly  owing  to  these  factors 
that  it  is  impracticable  to  distribute  the  bacteria  in  liquid  cultures  and 
maintain  the  requisite  effectiveness. 

In  the  use  of  cultures  for  inoculating  soil  the  farmer  should  be 
guided,  as  in  all  other  matters  pertaining  to  soil  treatment,  by  his  own 
peculiar  needs  and  should  not  give  too  great  weight  to  the  experiences 
of  others  whose  soil  conditions  may  differ  widely.  It  would  be  unwisi 
to  invest  largely  in  any  new  method  for  increasing  plant  growth, 
whether  bacterial  or  of  any  other  nature,  i •p/'thout  previously  experi- 
menting in  a  small  way. 

DANGER  OF  INOCULATION  BY  SOIL  TRANSFER. 

Satisfactory  inoculations  have  been  obtained  by  transferring  soil 
from  old  fields  on  which  the  legume  has  been  grown,  but  experience 
has  shown  that  there  are  dangers  incident  to  such  methods  of  soil 
transfer  which  it  is  wise  to  avoid. 

The  source  of  supply  of  such  soil  should  be  very  definitely  known, 
and  in  no  case  should  soil  be  used  from  fields  which  have  previously 
borne  any  crops  affected  with  a  fungous  disease,  a  bacterial  disease, 
or  with  nematodes.  Where  a  rotation  of  crops  is  practiced,  it  is  often 
difficult  to  make  sure  of  this  factor,  so  that  the  method  of  soil  transfer 
is,  under  average  circumstances,  open  to  suspicion,  if  not  to  positive 
objection.  Numerous  animal  and  plant  parasites  live  in  the  soil  for 
years,  and  are  already  established  in  so  many  localities  that  it  is  mani- 
festly unwise  to  ship  soil  indiscriminately  from  one  portion  of  the 
country  to  another. 

The  bacterial  diseases  of  the  tomato,  potato,  and  eggplant,  and  the 
club-root,  brown-rot,  and  wilt  disease  of  the  cabbage,  all  more  or  less 
widely  distributed,  are  readily  transmitted  in  the  soil;  while  in  the 
South  and  West  there  are  the  wilt  diseases  of  cotton,  melons,  sweet 
potatoes,  cow  peas,  and  flax,  and  various  nematoid  and  root-rot  diseases 
which  might  easily  become  a  serious  menace  over  areas  much  larger 
than  they  now  occupy  if  deliberately  spread  by  the  careless  use  of 
soil  for  inoculation  purposes.  There  are  several  insect  and  fungous 
diseases  of  clover  to  be  avoided,  and  various  diseases  of  beans  and 
peas.     There  is  also  a  disease  of  alfalfa,  the  ••leaf  spot.""  which  is 


IT  Kl   ■<    I    I.I  I    Rl      [NOOl    |.  \TlnN.  9 

causing  damage  in  some  regions.  These  arc  only  a  few  of  many  dis 
eases  liable  to  be  transmitted  in  soils.  The  farmer  should  therefore 
be  on  his  guard.  The  danger  from  such  sources  is  l>\  no  means 
imaginary.  The  Department  of  Agriculture  has  had  specific  cases  of 
such  accidental  distribution  reported,  and  if  the  business  of  selling 
soil  tor  inoculation  is  made  to  Sourish  by  farmers  purchasing  without 
question  "alfalfa  -oil."  "cowpea  soil,"  etc.,  there  is  every  reason  to 
believe  thai  experience  will  demonstrate  the  folly  of  such  haphazard 
methods. 

Of  scarcely  less  importance  is  the  danger  of  disseminating  noxious 
weeds  and  insect  pests  through  thi^  plan  of  inoculation  by  mean- of 
Boils.  Even  though  weeds  may  not  have  been  serious  in  the  first  field, 
tlic  great  numbers  of  dormant  seeds  requiring  but  a  slight  change  in 
surroundings  to  produce  germination  are  always  a  menace.  The  enor- 
mous damage  to  crops  caused  by  introduced  insects  and  weeds  should 
convey  a  warning  and  lead  to  caution.  It  is  not  the  part  of  good 
judgmenl  to  view  the  risk  as  a  slight  one  justified  l>\  the  end  in  view. 

PURE-CULTURE  INOCULATION. 

The  extensive  experiments  carried  on  by  the  Department  of  Agri- 
culture during  L904  demonstrated  the  fact  that,  by  the  proper  use  of 
pure  cultures,  the  nodule  bacteria  are  actually  carried  into  the  soil  in 
such  a  way  a-  to  form  root  nodules,  and  where  other  condition-  are 
favorable  the  inoculation  thus  brought  about  makes  possible  the 
growth  of  each  legume  in  soils  where  it  had  previously  failed  from 
the  lack  of  bacteria.  The  original  cultures  used,  however,  must  be 
prepared  with  the  utmost  care  and  with  a  view  to  preserving  and 
increasing  their  natural  power  as  "  nitrogen  fixers"  rather  than  merely 
to  make  them  grow  under  favorable  conditions.  The  methods  devised 
in  our  Laboratory  of  Plant  Physiology  are  based  on  well-recognized 
principles  of  plant  breeding  and  -election,  and  mark  a  decided  advance 
in  the  production  of  culture-  for  soil  inoculation.  The  old  pure- 
culture  method-  were  not  effective,  for  reasons  clearly  stated  by 
Dr.  Moore  in  Bulletin  No.  71  of  the  Bureau  of  riant  Industry  and 
by  Dr.  Moore  and  Mr.  Robinson  in  Farmers'  Bulletin  No.  21  t. 

The  Department  of  Agriculture  is  continuing  the  work  of  develop- 
ing types  of  the  bacteria  associated  with  leguminous  plants,  which 
will  have  greater  activity,  collecting  from  the  air  more  nitrogen  per 
acre  than  forms  now  common  in  nature  or  available  from  laboratories. 
It  i-  desirable  that  similar  investigations  should  be  conducted  with 
reference  to  the  nitrogen-fixing  bacteria  existing  in  the  soil  independ- 
ent of  the  legumes.  Important  steps  have  already  been  taken  along 
this  line,  but  the  very  large  demand  for  cultures  for  leguminous  crops, 
by  consuming  the  time  ^>\'  the  laboratory  force,  has  seriously  retarded 
these  investigations  during  the  past  year. 


10  NITROGEN-FIXING    BACTEKIA. 

The  Department  is  reacty  to  cooperate  with  experiment  .stations  and 
commercial  firms,  to  give  and  to  receive  suggestions,  to  test  the  prod- 
uct of  others,  and  to  furnish,  as  far  as  possible,  cultures  to  be  tested 
in  the  laboratory  and  under  field  conditions. 

There  is  nothing  in  the  nature  of  the  processes  involved  which 
would  prevent  a  competent  bacteriologist,  after  some  experience  in 
this  particular  field,  from  producing  cultures  of  as  high  a  grade  as 
those  sent  out  by  the  Department,  and  every  assistance  will  be  given 
to  competent  persons  desiring  to  undertake  the  work. 

A.  F.  Woods, 
Acting  Chief,  Bureau  of  Plant  Industry. 
Approved: 

James  Wilson, 

Secretary  of  Agriculture. 

Washington,  D.  C,  May  6,  1905. 


I 


*3 


* 


* 


UNIVERSITY  OF   FLORIDA 


3  1262  08928  3286 


